E. coli MutY and its human homologue (MUTYH, formerly MYH) play an important role in the prevention of mutations associated with 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) by removal of misincorporated adenine residues from OG:A mismatches. This glycosylase has garnered the spotlight by the discovery of a correlation between inherited MUTYH variations and colorectal cancer (CRC). Using pre-steady kinetics and noncleavable 2'-deoxyadenosine analogue-containing oligonucleotides, we provided information on the functional defects of two missense variants of MUTYH found in colorectal cancer that was key for establishing the connection between MUTYH variants and colorectal cancer (referred to a MUTYH-associated polyposis or MAP). The understanding of this predisposition mechanism is still at an early stage. The clinical data is emerging rapidly, and new mutations in MUTYH continue to be uncovered. However, at this point, it is not clear whether all of the MUTYH variations affect the adenine glycosylase activity, or result in reduced OG:A repair for other reasons. We propose to further our understanding of the relationship between MUTYH and colorectal cancer, as well as continue our understanding of the complex features of mismatch recognition by this unique base-excision repair glycosylase. Our underlying hypothesis is that a detailed molecular understanding of OG:A mismatch recognition goes hand-in-hand with revealing intricacies of the relationship between MUTYH and colorectal cancer. Specifically, we will develop cellular assays to reveal features that affect recognition and repair of OG:A mismatches by MUTYH. This will involve developing several assays to assess the repair of OG:A mismatches of MutY and MUTYH in E. coli and mammalian cell lines. We will also examine the effects of alterations of the damaged substrate on MUTYH-mediated repair. In addition, we will gauge the response of these cell based assays by evaluating specific catalytic and binding defects in MUTYH. We will also examine the importance of post-translational and interactions with protein partners on the activity of MUTYH using adenine glycosylase assays, mismatch binding experiments and cellular repair assays. MUTYH expressed in baculovirus-infected insect cells is phosphorylated and we will identify the locations of these phosphorylation sites in MUTYH. We will prepare the relevent phospho-mimetic and phospho-ablating mutations in MUTYH and analyze the adenine glycosylase activity and mismatch binding affinity of the mutated enzymes We will determine the consequences of MUTYH variations found in colorectal cancer on the instrinsic enzymatic properties, ability to mediate OG:A repair and prevent mutations in cells. This will include measuring the adenine glycosylase activity, mismatch affinity, the ability to mediate OG:A repair in both bacterial and mammalian cellular assays, the genomic mutation rate and sensitivity of cells expressing MUTYH variants to oxidative stress. This information will provide information as to whether a variant MUTYH is dysfunctional, and the origin of the dysfunction.